HB-EGF was isolated and purified by Higashiyama et al, in 1992 from a culture supernatant of a macrophage-differentiated human macrophage-like cell line U-937 (Non-patent document 1). HB-EGF holds 6 cysteine residues in common preserved in the epidermal growth factor (EGF) family and belongs to the EGF family, and is synthesized as a type I membrane protein similar to the case of other proteins belonging to the EGF family (Non-patent documents 1 and 2). The membrane type HB-EGF is converted into a secretory HB-EGF of 14 to 22 kilo daltons (hereinafter referred to as “kDa”) by a metalloprotease activated by various physiological stimuli such as stress due to heat or osmotic pressure, a growth factor, a cytokine and lysophosphatidic acid (LPA) which is a G protein coupled receptor (GPCR) (Non-patent documents 1 to 3). The secretory HG-EGF binds to an EGF receptor (EGFR/ErbB1) (Non-patent document 1), ErbB4 (Non-patent document 4) and N-arginine dibasic convertase (Non-patent document 5), and has the growth acceleration activity for fibroblasts and smooth muscle cells (Non-patent document 1), keratinocyte (Non-patent document 6), hepatocyte (Non-patent document 7) and mesangial cell (Non-patent document 8). In addition, it is also known that HB-EGF is related to organogenesis of, for example, cardiac valve (Non-patent documents 28, 29 and 31), healing of wound (Non-patent documents 9 and 10), hyperplasia of smooth muscle cell caused in atherosclerosis (Non-patent document 11), re-stricture (Non-patent documents 12 and 13), pulmonary hypertension (Non-patent document 14), hepatic regeneration (Non-patent document 15), cerebral disorder (Non-patent document 16) and cancer (Non-patent documents 28 to 35).
On the other hand, it has been reported that a considerable amount of membrane type HB-EGF is expressed on the cell surface without being digested into its secretory (Non-patent document 17). It is known that the membrane type HB-EGF forms a complex on the cell surface with CD9 or the like tetra tetraspanin or integrin α3β1, and it has been reported also that it interacts as a juxtacrine growth factor with adjacent cells (Non-patent documents 17 to 22). In addition, Naglich et al. have reported that the membrane type HB-EGF functions as receptor of diphtheria toxin and is related to the internalization of diphtheria toxin into cells (Non-patent document 23).
When Mekada et al. have analyzed physiological functions of HB-EGF by preparing HB-EGF knockout (KO) mice, the HB-EGF KO mice showed dilation of ventricle, lowering of cardiac function and a symptom of cardiac valve hypertrophy and more than half of the animals died in several days after birth. This fact shows that HB-EGF is a protein essential for the development and functional maintenance of the heart (Non-patent document 24).
Next, Mekada et al. have prepared two genes for an HB-EGF which became unable to be converted into secretory due to introduction of a mutation into a protease digestion site (hereinafter referred to as “HBuc”) and an HB-EGF which lacks a transmembrane region, is secreted and is secreted independently of protease digestion (hereinafter referred to as “HBΔtm”). By preparing transgenic mice which express respective HB-EGF mutants, physiological functions of membrane type and secretory HB-EGFs were analyzed (Non-patent document 25). As a result, since the HBuc expressing mice showed symptoms similar to those of the HB-EGF KO mice, it was considered that the secretory HB-EGF is functioning as the active type protein. Most of the HBΔtm expressing mice died before the neonatal stage or at the neonatal stage. In addition, hyperplasia of keratinocyte and ventricular hypertrophy from the neonatal stage were found in HBΔtm/+ expressing mice in which a mutation was introduced into only one of the alleles. These symptoms were phenotypes directly opposite to those of the HB-EGF KO mice and HBuc mice. CRM197 known as a mutant of diphtheria toxin (Non-patent document 26) specifically inhibits cell growth acceleration activity of HB-EGF and does not permeate cell membrane. Since this CRM197 inhibited hyperplasia and ventricular hypertrophy as phenotypes of the HBΔtm expressing mice, it is considered that the HBΔtm formed in the HBΔtm expression mice does not act by binding to its intracellular receptor before its secretion, but acts by binding to the receptor on the cell surface after secreted extracellularly. Accordingly, the quantitative balance between membrane type HB-EGF and secretory HB-EGF in the living body is essential for the maintenance of normal physiological functions, and it is considered that the process for converting from membrane type into secretory of HB-EGF is controlled in the living body.
Higashiyama et al. have found that secretory HB-EGF protein in the heart is increased in the heart of a mouse in which cardiac hypertrophy was induced by constricting the thoracic aorta. It has been reported that when a low molecular weight compound capable of inhibiting a protease which converts membrane type HB-EGF into secretory is administered to this mouse, cardiac hypertrophy is suppressed as a result of suppressing conversion of the membrane type HB-EGF into secretory in the heart (Non-patent document 27).
It has been reported so far that HB-EGF is expressed at a high level in various cancers such as beast cancer, liver cancer, pancreas cancer and bladder cancer, in comparison with normal tissues (Non-patent documents 28 to 31). Also, it has been recently found that HB-EGF is an important factor for the proliferation of cancer (Non-patent documents 32 and 33). Mekada et al. have found that a significant tumor growth inhibitory effect is recognized when small interference RNA (siRNA) of HB-EGF is introduced into a cancer cell line, or CRM197 is administered to a mouse into which the cancer cell line was transplanted, in a model system in which a human ovarian cancer cell line is transplanted into a nude mouse. Also, Higashiyama et al. have found that cell growth, colony forming ability, vascular endothelial growth factor (VEGF) expression and expression of cyclin D1 and the like are increased in vitro in a bladder cancer cell line into which the HB-EGF gene was transferred. In addition, it was reported that increase of tumorigenicity and increase of tumor angiogenesis are found also in vivo. Such a growth stimulation activity was found only when the membrane type HB-EGF gene or secretory HB-EGF gene was expressed, but was nor found when a protease-resistant membrane type HB-EGF gene was forcedly expressed. Accordingly, a possibility was suggested that the secretory HB-EGF is an important factor which is related to the tumor growth of ovarian cancer and bladder cancer. Regarding the expression of HB-EGF in clinical patients, Mekada et al. have analyzed expression quantity of HB-EGF mRNA and concentration of secretory HB-EGF in the tumor tissues and ascites of ovarian cancer patients, and reported that only HB-EGF among the EGF family is expressed (Non-patent document 32). In addition, Miyamoto et al. have reported that prognosis is poorer in ovarian cancer patients in which HB-EGF mRNA of the tumor is highly expressed, than low expression patients (Non-patent document 34). The above results show that at least in the ovarian cancer, the secretory HB-EGF produced by the cancer is related to the cancer growth by the autocrine or paracrine mechanism (Non-patent document 35). As antibodies which bind to secretory HB-EGF and inhibit its activity, some polyclonal antibodies and one monoclonal antibody (all manufactured by R & D) are known. It has been reported that an anti-HB-EGF goat polyclonal antibody (manufactured by R & D) binds to the cell surface membrane type HB-EGF expressed in COS-7 cell (Non-patent document 3). It is broadly known that when a membrane protein is present on the surface of a cell such as cancer, a monoclonal antibody which binds to such a protein could become a therapeutic agent which inhibits growth of the cell (Non-patent document 36). However, there are no reports to date for monoclonal antibodies which bind to a secretory HB-EGF, a cell membrane-bound HB-EGF and a membrane type HB-EGF.    Non-patent document 1: Science, Vol. 251, 936, 1991    Non-patent document 2: J. Biol. Chem. 267 (1992) 6205-6212    Non-patent document 3: Nature, Vol. 402, 884, 1999    Non-patent document 4: EMBO J. 16 (1997) 1268-1278    Non-patent document 5: EMBO J. 20 (2001) 3342-3350    Non-patent document 6: J. Biol. Chem. 269 (1994) 20060-20066    Non-patent document 7: Biochem Biophys. Res. Commun. 198 (1994) 25-31    Non-patent document 8: J. Pathol. 189 (1999) 431-438    Non-patent document 9: Proc. Natl. Acad. Sci. U.S.A. 90 (1993) 3889-3893    Non-patent document 10: J. 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